Platelet Reactivity of Aspirin and Clopidogrel in Coronary Artery Disease Patients with or Without Diabetes Mellitus

Available online at www.ijpcr.com International Journal of Pharmaceutical and Clinical Research 2016; 8(7): 690-694 ISSN- 0975 1556 Research Article ...
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Available online at www.ijpcr.com International Journal of Pharmaceutical and Clinical Research 2016; 8(7): 690-694 ISSN- 0975 1556 Research Article

Platelet Reactivity of Aspirin and Clopidogrel in Coronary Artery Disease Patients with or Without Diabetes Mellitus Suharjono1*, Wahyudi E D1, Yunita A1, Yogiarto M2, Zulkarnain B S1 1 Department of Clinical Pharmacy, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia Department of Cardiology, Faculty of Medicine, Airlangga University / Dr Soetomo Teaching Hospital, Surabaya, Indonesia

2

Available Online:15th July, 2016 ABSTRACT Background: Antiplatelet combination Aspirin (ASA) and Clopidogrel (CPG) has become part of therapeutic standard in patients with coronary artery disease (CAD). Thus, a decline in antiplatelet response due to platelet functional problem may be occured among CAD patients with diabetes mellitus (DM). Objective: To evaluate ASA platelet reactivity (Aspirin Reactivity Unit=ARU) and CPG (Platelet Reactivity Unit=PRU) among CAD patients with vs without DM who take a combination therapy of ASA 100 mg and CPG 75 mg/day. Methods: The study was done at Cardiology Outpatient Clinic - Dr. Soetomo Teaching Hospital Surabaya. A cross sectional observational study was carried out and an unpair qualitative analysis study was used to interprete the data. Platelet functional test was performed by VerifyNow, which was very specific for ASA (Aspirin Reactivity Unit = ARU) and CPG (Platelet Reactivity Unit = PRU). Results: A total of 20 patients were included in the study with the proportion of 10 CAD patients with DM and 10 CAD patients without DM. For ARU, about 4 CAD patients (40%) with DM and 3 CAD patients (30%) without DM seemed to have a resistance on ASA (p=1.000). For PRU, there were High On-Treatment Platelet Reactivity (HPR) on CAD patients with DM compared to CAD patients without DM (77.8% vs 11.1%; p = 0.015). Conclusion: Eventhough the number of CAD patient with DM who showed resistance to ASA was slightly higher compared to CAD patient without DM, there was no statistical difference between two groups. But for CPG, there were very significant higher HPR on CAD patient with DM than without DM. Keywords: Aspirin, Clopidogrel, Type 2 Diabetes Mellitus, Coronary Artery Disease, Platelet Reactivity INTRODUCTION Coronary Artery Disease (CAD) is a sort of heart disease caused by abnormalities in the coronary arteries. Generally, Diabetes Mellitus (DM) has a very important impact on atherosclerosis. According to its natural history, the process of type II DM is accompanied by increased of platelet hyperactivity, thromboxane level in the blood vessels that facilitates the aggregation of platelets, coagulation factors and platelet function abnormalities, as well as the increased of intracellular calcium. In another side, there is decrease in the production of nitric oxide, platelet turnover following platelet structural damage caused by hyperlipidemia, and fibrinolysis proccess that finally contribute to an increase in thrombotic process and the development of atherosclerosis1. Platelet aggregation plays an important role in the development of ischemia in CAD patients and thromboischemic complication after Percutaneous Coronary Intervention (PCI). Platelet activation mediates the inflammation at blood vessels’ walls that leads to vessel damage after platelet aggregation and the generation of thrombin2. Aspirin (ASA) and Clopidogrel (CPG) are major antiplatelet agents in atherosclerosis. Both of these drugs are often together given because theit

*Author for Correspondence

better effectiveness. This study aimed to evaluate the reactivity of ASA and CPG in patients receiving both ASA and CPG. This is the first study to investigate ASA and CPG Resistance conducted in Indonesian patients with or without diabetes. The number of patients who consume either ASA, CPG or both in our country is very high. Hence, it is interesting for our health care team to initiate pilot study to investigate this phenomenon. MATERIAL AND METHODS This was an analytic observational study with cross sectional design. Selection of study subjects began in May 2014 - July 2014 with consecutive sampling. Inclusion criterias were CAD patients with and without type 2 DM, age > 20 years old, had received 100 mg ASA therapy and 75 mg CPG daily for 7 days, and willing to participate in research as written in the informed consent. Then, the exclusion criterias were patients with history of gastrointestinal disorders, platelet count 2 mg / dl, and heart

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Table 1: Patients Demography Variables Total Patients with CAD (n=20) with DM (n=10) without DM (n=10) Sex Male 5 (50%) 7 (70%) Female 5 (50%) 3 (30%) Age (Years) 35-40 1 (10%) 1 (10%) 41-50 1 (10%) 3 (30%) 51-60 4 (40%) 4 (40%) 61-70 3 (30%) 1 (10%) 71-80 1 (10%) 1 (10%) Mean 55,6±11,3 58,4±7,5 BMI (kg/m2) 22.8 ± 9.7 25.3 ± 10,7 Comorbids Hypertension 1 (80%) 4 (40%) Hyperlipidemi 1 (60%) 1 (10 %) a 7 (70 %) 5 (50 %) Hypertension 1 (10 %) 0 (0 %) +Dyslipidemia None Hyperte nsion+ Dyslipi demia Risk Factors Smoking 2 (20%) 2 (20%) Drugs Used CPG 10 (100%) 10 (100%) Statin 8 (80%) 7 (70%) ACEI 8 (80%) 7 (70%) ARB 4 (40%) 2 (20%) CCB 6 (60%) 5 (50%) Nitrate 7 (70%) 6(60%) ß-Blocker 10 (10%) 8 (80%) Diuretic 4 (40%) 1 (10%) OAD 4 (40%) 0% Insulin 6 (60%) 0% failure were not included in the study. The parameters of ARU (Aspirin Reactivity Unit) and PRU (Platelet Reativity Unit) had been applied in the study by using VerifyNow. The patients’ blood sample was inserted into a vacuum tube containing platelet agonist (arachidonic acid in 3.2% sodium citrate) as its platelet function would be further evaluated. The values obtained in the form of infrared absorption resulted from the process of platelet agglutination. If the value of ARU > 550, it was said to be resistant to ASA. High On-Treatment Platelet Reactivity (HPR) was defined by the value of PRU > 208 and Normal On-Treatment Platelet Reactivity (NPR) PRU value ≤ 208. This research had passed the ethnical clearance from the ethic committee of Dr. Soetomo Teaching Hospital Surabaya. Distributions of variables were assessed for normality using Shapiro-Wilk test. ASA CPG resistance difference between CAD patients with and without diabetes type 2 were analyzed using chi square test. If the chi square test requirements are not

met, Fisher exact test was done. The result is statistically significant is p value more than 0.05. RESULTS Table 1 shows the demographic data of patients. There were 12 men and 8 women with a mean age of 57.2 ± 11.5 years in CAD group with type 2 DM and 53.4 ± 9.8 years in the CAD group without type 2 DM. The average BMI in the CAD group with type 2 DM was 22.8 ± 9.7 and 25.3 ± 10.7 in the group without type 2 DM. In both groups, hypertension was the most comorbidities, 8 patients in the CAD group with type 2 DM and 9 in the CAD group without type 2 DM. The number of patients with hyperlipidemia was equal between two groups. Figure 1, 2 and Table 2 present ARU from 10 CAD patients with DM (mean 497.00 ± 126.044) and 10 CAD patients without DM (mean 469.20 ± 109.381). There were aspirin resistance (ARU > 550) among 4 CAD patients with DM and 3 CAD patients without DM. Thus, no significant difference found between groups (p=1.000). Figure 1, 2 and Table 3 present PRU from 9 CAD patients with DM and 9 CAD patients without DM. There were High Platelet Reactivity on CPG (PRU > 208) among 8 CAD patients with DM (mean 228.44 ± 68.672) and 2 CAD patients without DM (mean 145.33 ± 42.971). Thus, statistically significant difference found between groups (p=0.015). DISCUSSION From the study, it can be analyzed that 4 CAD patients with type 2 DM and 3 CAD patients without type 2 DM show Aspirin resistance. Thus, based on statistical analysis using Fisher exact Test, it can be said that the difference is not statistically significant between two groups (p = 1.000). This non-significancy may be caused by other factors or comorbidities such as age, Body Mass Index (BMI), smoking, and genetic3. The average age in CAD group with DM is 57.2 ± 11.2 years old, while in CAD group without DM was 53.4 ± 9.8 years old. In the study of ASA resistance conducted by Vatury3 to 583 patients who received a dose of 75-325 mg per day, an increase in the value of ARU > 550 was shown in those aged > 75 years old (p = 0.007). Contrary, there was nonsignificant increase in the value of ARU among those aged < 75 years old (p = 0.40). The influence of age on ASA resistance might be due to the higher level of microvascular disturbances in the advanced age which lead to the decrease of nitric oxide synthesis and disruption of platelet response to vasoactive substances4,5. In both groups of this study, hypertension and dyslipidemia still be two dominant risk factors. High plasma glucose level would lead to the increase level of Reactive Oxygen Species (ROS) and Advanced Formation Glycosylation End Product (AGE Product) at endothelial blood vessels which finally cause endothelial damage and inflammation of blood vessels. The inflammation of blood vessels will trigger the synthesis of nitric oxide, causing vasodilatation6. ASA resistance occurs more often among women with the statistical results of 34.4% vs. 17.3% (p = 0.001)7. Another study

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Figure 1: Value of ARU and PRU among 10 CAD patients with DM (note: Patients no. 10: PRU not available)

Figure 2: Value of ARU and PRU among 10 CAD patients without DM

NPR = normal platelet reactivity if PRU ≤ 208 HPR = high platelet reactivity if PRU > 208 Table 2: Statistical Analysis of ARU among CAD Patients with DM and without DM ARU / n = 10 DM without DM Mean ± SD 497.00 ± 126.044 469.20 ± 109.381 Sum of Patients Aspirin Resistance (ARU > 550) 4 (40%) 3 (30%) Aspirin Sensitive (ARU < 550) 6 (60%) 7 (70%) Incidence of aspirin sensitivity among CAD patients with DM and without DM were not statistically significant (p=1.000) conducted by Lev8 supported the finding that women tend to have ASA resistance than men (23.4% vs. 7.8%; p = 0.01). This phenomenon might be due estrogen protective effect against atherosclerosis. Besides, women are also more prone to disruption of vasodilation response to acetylcholine. In this study, the numbers of women who develop ASA resistance are less than male (3 vs 4 patients). In CAD patients group without type 2 DM who develop ASA resistance are those who smoke. In another side, the group of CAD patients with type 2 DM who smoke doesn’t show any ASA resistance. Unfortunately, the number of smoker in this study is not enough to detect the significant difference, but smoking plays role in ASA resistance. In acute conditions, smoking increases platelet thrombus formation at arterial wall. Inhibition of platelet

COX by ASA may not be sufficient in preventing acute conditions. An increase of platelet thrombus formation is due to the elevated aggregation response to thrombin. The acute increase of platelet thrombus formation after smoking may be associated with the higher levels of epinephrine to increase platelet aggregation although ASA therapy has been given9 to both groups in accordance with the CAD guidelines including the provision of β-blockers, statins, and ACEI10. In both of groups, some of CAD patients with type 2 DM were treated with a combination of antidiabetic drugs (OAD) and insulin. Insulin acts as inhibitor of platelet aggregation. It owns a protective effect against thrombus formation and release of vasoactive and chemotactic mediators which contribute to the pathophysiology of

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Table 3: Statistical Analysis of PRU among CAD Patients with DM and without DM PRU / n = 9 DM without DM Mean ± SD 228.44 ± 68.672 145.33 ± 42.971 Sum of Patients (%) HPR ( PRU > 208) 7 (77.8%) 1 (11.1%) NPR (PRU ≤ 208) 2 (22.2%) 8 (88.9%) Incidence of HPR among CAD patients with DM and without DM was statistically significant difference (p=0.015) thrombosis and atherosclerosis. A research conducted by Ariturk11 had described that ASA resistance occurred in OAD group by 4.7%, and 8.6% in the insulin group. There was no statistically significant difference between the OAD and insulin groups (p = 0.359). ASA resistance may also be caused by genetic variation. A study that supports individual variation in respond to ASA had been conducted by Shen12 on Heredity and Performed type Intervention (HAPI) Heart study. A research of 745 patients, with the proportion of 400 men and 345 women, was organized using the TAT (Thrombocytes Aggregation Test) with the dose of 81 mg / day ASA after patients taking ASA for 14 days. Thus, as many as 21% of patients showed ASA resistance, 30% of them were women and 16% of them were men. It might be caused by fewer barriers of platelet aggregation in women. ASA resistance may also be due to genetic polymorphism. Genetic polymorphisms in conjunction with ASA resistance is most commonly caused by several factors, or can also be caused by other hemostatic genetic variation (polymorphism in blood coagulation and fibrinolytic factors). A series of single nucleotide polymorphisms (SNPs) in the genes of prostaglandin endoperoxide synthase 1 (PTGS1) includes A842G, C22T [R8W], G128A [Q41Q], C644A [G213G], and C714A [L237M] associated with the response to ASA13. At the cellular level of platelets, ASA translocation to cytoplasm in Multidrug Resistance is influenced by the presence of protein-4 (MRP-4). An increase in MRP-4 will lead to the decrease of intracellular availability of ASA. The increase of MRP-4 is individualy varied and influenced by some diseases, such as DM. MRP-4 is generally elevated among patients after revascularization using coronary artery bypass grafting. Dipyridamole inhibits MRP-4. Therefore, it becomes the reason why ASA effects do increase after administration of dipyridamole14. In this study, platelet function has been assessed with the VerifyNow P2Y12 assay. Here we found that CAD patients with type 2 DM have higher platelet reactivity despite adequate CPG pretreatment compared to CAD patients without type 2 DM (Table 3) in agreement with previous research. Detailed studies have found that platelets from diabetics are generally more reactive and less responsive to antiplatelet 15,16. Several mechanisms are involved in this platelet dysfunction. These include hyperglycemia, insulin resistance, and increased inflammatory status 17. Hyperglycemia enhances platelet aggregation by inducing P-selectin expression, by activating protein kinase C (a mediator of platelet activation) and by glycating platelet surface proteins, with consequent decrease in membrane fluidity and amplification of platelet adhesion 18. Hyperglycemia also induces an increase in non-enzymatic

glycation of low-density lipoprotein (glycLDL) which renders platelets more susceptible to oxidative stress. Additionally, glycLDL may induce platelet dysfunction by increasing the intracellular calcium concentration and inhibiting platelet membrane sodium–potassium-ATP activity19,20. Moreover, diabetic patients with poor glycemic control have increased platelet reactivity despite dual antiplatelet therapy. Increased plasma fibrinogen is significantly associated with a lower response to CPG in patients with DM, possibly due to a direct interaction of fibrinogen with the glycoprotein IIb/IIIa receptor. Furthermore, in diabetic patients increased production of platelet agonists, such as epinephrine and thrombin receptor agonist peptide, may explain the higher levels of platelet activation through different signaling pathways besides those depending on the P2Y12 receptor15. Platelets are the target of insulin, which interacts with its own receptor on the surface of the platelet.This results in suppression of cAMP, inhibition of P2Y12 signalling and reduced platelet reactivity, however platelets of diabetic patients are victims of the insulin resistance phenomenon that characterises diabetes mellitus type 2 and results in decreased sensitivity to insulin. The end result is upregulation of the P2Y12 pathway and increased platelet reactivity17,20. Chronic hyperglycemia can result in reactive oxygen species (ROS) production directly via glucose metabolism and auto-oxidation, and indirectly through the formation of advanced glycation end products and their receptor binding. ROS may then activate signaling molecules within endothelial cells, including PKC and nuclear factor-kB (NF-kB), resulting in transcription of redox sensitive genes which switch to proinflammatory and prothrombotic phenotypes. ROS may also induce structural and functional changes in coagulative proteins. Finally, ROS may lead to the formation of 8-iso-PGF2a, a non-enzymatic oxidation product of circulating LDL and arachidonic acid, which mediates vasoconstriction and platelet hyperreactivity. Not surprisingly, improved glycemic control is associated with a significant reduction in both lipid peroxidation and platelet activation in vivo19. Other mechanisms linked to HTPR include increased exposure to ADP, increased cytosolic level of calcium, and increased platelet turnover17. Thus, in patients with DM a global hyperreactive platelet status is present, which may explain low responsiveness even after higher maintenance doses of antiplatelet drugs21. CONCLUSION From the results of research on ASA resistance in CAD patients with and without type 2 DM that was conducted in Cardiology Outpatient Clinic Dr. Soetomo Public Hospital Surabaya from May to August 2014, it can be

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concluded that there is no statistically significant difference related to ASA resistance among patients with and without type 2 DM. Diabetic patients tend to have higher platelet reactivity compared to non-diabetic patient. This study suggests that the use of platelet function test may help to identify patients with HPR despite adequate CPG pre-treatment. Patients with DM

undergoing PCI need to get a more aggressive antiplatelet regimen to decrease the incidence of events after PCI.

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Implantation. Clin Appl Thromb Haem 2012; 18(3): 249-57. 3. Vatury M, Vaduganathan M, Bental T, Solodky A, Kornowski R. Relation of aspirin Response to Age in Patients With Stable Coronary Artery Disease. Am J Cardiol 2013; (11): 212-16.

ACKNOWLEDGMENTS Director of Dr. Soetomo Teaching Hospital and The Head of Cardiology and Vascular Medicine Department Dr.Soetomo Teaching Hospital for the research permission.

BD, Shuldiner AR. Aspirin Resistance in Healthy Drug-Naive Men Versus Women (from the Heredity and Phenotype Intervention Heart Study). Am J Cardiol 2009; 104:60612. 13. Maree AO, Curtin RJ, Chubb A, Dolan C, Cox D, O'Brien J, Crean P, Shields DC, Fitzgerald DJ. Cyclooxygenase-1 haplotype modulates platelet response to aspirin. J Thromb Haemost 2005; 3:23405. 14. Bultas J. Antiplatelet therapy. A Pharmacologist’s perspective 2013; 55:86-94. 15. Mangiacapra F. Comparison of Platelet Reactivity and Periprocedural Outcome in Patients With Versus Without Diabetes Mellitus and Treated With Clopidogrel and Percutaneous Coronary Intervention. J Am Coll Cardiol 2010; 106:619-23. 16. Kirtane AJ. Impact of Point of Care Platelet Function Testing Among Diabetic and Non-Diabetic Patients Undergoing PCI with Drug-Eluting Stent: an ADAPTDES Substudy. J Am Coll Cardiol 2012; 59(13):E268. 17. Ait-Mochtar O, Bonello L, Benamara S, Paganelli F. Hight on Treatment Platelet Reactivity. Heart Lung Circ 2012; 21(1):12-21. 18. Patti G, Proscia C, Sciascio GD. Antiplatelet Therapy in Patients with Diabetes Mellitus and Acute Coronary Syndrome: New Insights from Randomized Trials. Circulation 2014; 78:33-41. 19. Angiolillo DJ, Suryadevara S. Aspirin and Clopidogrel: Efficacy and resistance in Diabetes Mellitus. Best Pract Res Clin Endocrinol Metab 2009; 23(1):375-88. 20. Ferreiro JL, Angiolillo DJ. Diabetes and Antiplatelet Therapy in Acute Coronary Syndrome. Circulation 2011; 123(1):789-813. 21. Angiolillo D, Shoemaker SB, Desai B. Randomized comparisson of a high clopidogrel maintenance dose in patients with diabetes mellitus and coronary artery disease: result of the Optimizing Antiplatelet Therapy in Diabetes Mellitus (OPTIMUS) study. Circulation 2007; 115:708-16.

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